Building on decades of work showing how T cell receptors recognize peptide antigens bound to MHC proteins, the discovery of the CD1 antigen presentation system shows that T cells also respond to changes in cellular lipids. During the first phase of this proposal, the applicant has discovered phosphoisoprenoid antigens presented by CD1c proteins, determined the enzymatic basis of their biosynthesis and found that they activate T cells derived from the lesions of human patients with autoimmune thyroiditis. These studies also identified two previously unknown classes of lipopeptide antigens presented by CD1c and CD1a, which use fattyl acyl chains to anchor to CD1 proteins, leading to presentation of their peptide moieties to T cell receptors. Recent advances in the synthesis of lipid antigens, along with crystal structures showing the precise dimensions and shapes of CD1 antigen binding grooves, now allow detailed investigation of the molecular interactions among CD1, lipid antigens and T cell receptors. The continuation proposal uses dolichyl glycolipids and acylpeptides as model antigens to determine the mechanisms by which CD1a and CD1c present a carbohydrate moiety and a peptide moiety to human T cell receptors in vitro and during the course of autoimmune thyroiditis. Using synthetic antigen analogs, we will determine the influence of carbohydrate linkage, lipid size and peptide length for binding to recombinant CD1 proteins. CD1-lipid complexes will be tested for their ability to bind to and activate T cell receptors in vitro. Mass spectrometric analysis of lipids eluted from cellular CD1 proteins will identify the molecular structures of endogenous lipids loaded onto CD1 proteins in cells. Last, existing and newly discovered lipid antigens will be tested for their ability to be recognized by polyclonal T cells from the thyroid glands and peripheral blood of human patients with Graves'disease and Hashimoto's thyroiditis. Relevance to public health. These studies provide a new model for understanding the molecular targets of T cells in a common human autoimmune disease, which is highly organ-specific. Further, determination of the specific size and chemical characteristics of lipids that bind to CD1 and activate T cells provides the necessary information for design of immunomodulatory lipids that can be applied as drugs to increase, decrease or polarize the functions of human T cells in vivo.